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By Pat Kyle on Thursday, August 19, 2004 - 09:22 am:
I'm having trouble with the in-gel tryptic digestion of cytochrome P450 enzymes. I am using ExPASy Peptidmass for peptide mass prediction, but I do not recognize any peaks upon infusing my digest extract. I am using electrospray MS (PE Sciex 365). Am I not concentrating my sample enough? Is desalting absolutely necessary?
My protocol is as follows:
1) Excise gel bands and chop into 1mm cubes
2) Destain using MeOH/H2O/HAC (45:45:10)
3) Shrink gel cubes with 100% acetonitrile
4) Allow cubes to swell in 12 ng/uL Trypsin Sol
5) Allow to incubate overnight at 37 degrees C
6) Vortex with Acetonitrile/H2O (50:50) to extract peptides
7) Vortex with Acetonitrile/50mM Ammonium Bicarb (50:50) to extract peptides
Pool the extracts (~300uL total)
9) Infuse onto MS/MS or inject onto LC/MS
I'm having trouble with the in-gel tryptic digestion of cytochrome P450 enzymes. I am using ExPASy Peptidmass for peptide mass prediction, but I do not recognize any peaks upon infusing my digest extract. I am using electrospray MS (PE Sciex 365). Am I not concentrating my sample enough? Is desalting absolutely necessary?
My protocol is as follows:
1) Excise gel bands and chop into 1mm cubes
2) Destain using MeOH/H2O/HAC (45:45:10)
3) Shrink gel cubes with 100% acetonitrile
4) Allow cubes to swell in 12 ng/uL Trypsin Sol
5) Allow to incubate overnight at 37 degrees C
6) Vortex with Acetonitrile/H2O (50:50) to extract peptides
7) Vortex with Acetonitrile/50mM Ammonium Bicarb (50:50) to extract peptides
Pool the extracts (~300uL total)
9) Infuse onto MS/MS or inject onto LC/MS
